The goal of this "high risk/high impact" project is to develop generally applicable methods which will allow solution NMR methods to be brought to bear upon the 3-D structures of integral membrane proteins (IMPs) of at least 20 kDa in molecular weight and having multiple transmembrane spans. The fundamental problems confronting NMR studies of larger membrane proteins are two-fold: (1) the use of classical detergents to solubilize complex IMPs inevitably leads to a large increase in effective molecular weight due to the association of 10s of kDa of detergent and (2) conditions which do lead to sharp NMR resonances typically also unfold the IMP (e.g., solubilization with organic solvents or examination of micellar IMPs at very high temperatures). The aims of this proposal are to develop sample preparation methods which would allow these problems to be overcome. Specifically: Aim 1. Amphipathic polymers "amphipols" will be employed as a possible route to solubilizing membrane proteins in aqueous solutions without greatly increasing the effective molecular weight of the protein. Both existing and novel amphipols will be tested. Aim 2. Chemical cross-linking methods will be examined as a way of conferring conformational stability to IMPs so that they can be examined under conditions which promote spectral quality, but which would ordinarily result in protein unfolding. These aims are interconnected. IMPs which will be used in these studies are E. coli diacylglycerol kinase and bacteriorhodopsin.